Human Jak2 kinase

ABSTRACT

The present invention provides a polynucleotide (hjak2) which identifies and encodes a novel human Jak2 kinase (HJAK2) which was expressed in the placenta. The present invention also provides for antisense molecules and oligomers designed from the nucleotide sequence or its antisense. The invention further provides genetically engineered expression vectors and host cells for the production of purified HJAK2 peptide, antibodies capable of binding to HJAK2, inhibitors which bind to HJAK2 and pharmaceutical compositions based on HJAK2 specific antibodies or inhibitors. The invention specifically provides for diagnostic assays based on altered hjak2 expression and which allow identification of such a condition. These assays utilize probes which comprise oligomers, fragments, or portions of hjak2 or its regulatory elements or antibodies specifically binding HJAK2.

This application is a divisional application of U.S. application Ser.No. 09/196,480, filed Nov. 19, 1998, now U.S. Pat. No. 6,019,966 whichis a divisional of U.S. application Ser. No. 08/567,508, filed Dec. 5,1995, issued Jun. 22, 1999, as U.S. Pat. No. 5,914,393.

FIELD OF THE INVENTION

The present invention relates to a novel, human Jak2 kinase isolatedfrom human placenta and to the use of this novel protein and its nucleicacid sequence in the diagnosis, study, prevention, and treatment ofdisease.

BACKGROUND OF THE INVENTION

JAK kinases are Janus family nonreceptor protein-tyrosine kinases(NR-PTK) that lack transmembrane regions and form functional complexeswith the intercellular regions of other cell surface receptors. Theywere first identified as the products of mutant oncogenes in cancercells where their activation was no longer subject to normal cellularcontrols. Described JAK kinases include Jak1, Jak2, and Jak3, which allshare the conserved kinase domain. In addition, these proteins have 5 to100 amino acid residues located on either side of, or inserted intoloops of, the carboxyterminal kinase domain which allow the regulationof each kinase as it recognizes and interacts with its target protein.Known target proteins include growth hormone receptor, prolactinreceptor, erythropoietin receptor, cytokine receptors and others whichutilize the common chain known as gp130. These receptors are unique bothin their ability to recruit multiple PTKs and in the diversity of theirresponses within different cell types (Taniguchi T (1995) Science268:251–55). Genetic evidence places these kinases in the interferon αand γ signal transduction pathways which are widely expressed inmammalian cells.

Kinases regulate many different cell proliferation, differentiation, andsignaling processes by adding phosphate groups to proteins. The highenergy phosphate which drives activation is generally transferred fromadenosine triphosphate molecules (ATP) to a particular protein by thePTKs, and the transfer process is roughly analogous to turning on amolecular switch. When the switch goes on, the kinase activates ametabolic enzyme, regulatory protein, receptor, cytoskeletal protein,ion channel or pump, transcription factor, or another kinase. Forexample, in their normal role, the JAK NR-PTKs are capable of regulatingtyrosine phosphorylation of STAT proteins, signal transducers andactivators of transcription, such that they translocate to the nucleusand bind DNA (David M et al. (1995) Science 269:1721–1723). In contrast,uncontrolled kinase signaling has been implicated in inflammation,oncogenesis, arteriosclerosis, and psoriasis.

Almost all kinases contain a similar 250–300 amino acid catalyticdomain. The N-terminal domain generally folds into a two-lobed structureto bind and orient ATP (or GTP) donor molecules. The larger C terminallobe binds the protein substrate and carries out the transfer ofphosphate from ATP to the hydroxyl group of a tyrosine residue. Theprimary structure of the kinase domain is conserved in the residues: G₅₀and G₅₂ in subdomain I, K₇₂ in subdomain II, G₉₁ in subdomain III, E₂₀₈in subdomain VIII, D₂₂₀ and G₂₂₅, in subdomain IX, and the amino acidmotifs of subdomain VIB, VIII and IX (Hardie G and Hanks S (1995)Academic Press, San Diego Calif.).

The novel human Jak2 kinase, hjak2, of the present application showssignificant conservation of the diagnostic kinase residues which allowedits identification from among the isolated cDNAs of a placenta library,the anatomy and physiology of which is briefly described below.

The placenta is a thickened disk-shaped temporary organ thatinterchanges gases, nutrients, hormones, excretory products, humoralantibodies (IgG), and any other circulating substances between thematernal and fetal bloodstreams. Receptors facilitate the transport ofglucose, amino acids, and IgG directly from maternal blood to fetalblood. The placenta is the only organ composed of cells derived from twoindividuals, the fetal extraembryonic chorion and the maternalendometrium. The boundary between these two tissues is marked byextracellular products of necrosis referred to as fibrinoid. Thisboundary results from the various tissue interactions, immunologicalresponses, etc. which occur in the placenta.

The major tissue interaction involves the expression of paternalantigens by the chorionic villi which is directly adjacent to maternalblood. Although the mother initiates an immunological response, fetaltissue is not typically rejected. This is attributed to the fact thatthe fetus only expresses major histocompatibility complex (MHC) I, andnot MHC II which is the major cause of organ allograft rejection. Inaddition, uterine secretions during early gestation contain significantamounts of glucose and glycoproteins which may participate in localimmunosuppression. Although infections by bacteria, viruses,mycoplasmas, or parasites may ascend from the endocervical canal orreach the placenta through maternal blood, they rarely cause grosspathological changes because of maternal immune defense.

Soon after implantation, fetal villi begin to control maternalphysiology to create an optimal environment for development. Thisinvolves the production of chorionic gonadotropin, estrogen andprogesterone, chorionic somatomammotropin, insulin-like growth factors,platelet derived growth factor, prolactin, and various cytokines. Theseand other factors such as hjak2 certainly regulate the numerousactivities (respiratory, immunological, gastrointestinal, and urinary)which occur within the placenta and between maternal and fetal tissues.

The anatomy and physiology of human placenta is reviewed, inter alia, inBenirschke and Kaufmann, (1992) Pathology of the Human Placenta,Springer-Verlag, New York N.Y., pp. 542–635; Herrera Gonzalez andDresser (1993) Dev Comp Immunol 17(1):1–18; Mitchell et al. (1993)Placenta 14:249–275; Naeye (1992) Disorders of the Placenta, Fetus, andNeonate; Diagnosis and Clinical Significance, Moseby Year Book, St.Louis Mo.; and Rutanen (1993) Ann Med 25:343–347.

SUMMARY

The present invention relates to a novel human Jak2 kinase and to theuse of the protein and its nucleic acid sequence in the study,diagnosis, prevention, and treatment of diseases. Human Jak2 kinase(hjak2) was first identified as a partial nucleotide sequence in IncyteClone 179527 during a computer search for nucleotide sequence alignmentsamong the cDNAs of a placenta library. A modified XL-PCR procedure,specially designed oligonucleotides, and cDNAs of the placenta librarywere used to extend Incyte Clone 179527 to full length. The assemblednucleotide sequence (SEQ ID NO: 1) hjak2 encodes the polypeptide (SEQ IDNO: 2) HJAK2 (SEQ ID NO: 2). Computer search and alignment of the fulllength amino acid sequence showed that HJAK2 has 92% similarity tomurine Jak2 kinase (MUSPTK1; GenBank GI 409584; Wilks A F (1989) ProcNat Acad Sci 86:1603–7), which in turn has 96% sequence similarity withhuman Jak1 kinase. These homologies and the conserved residues, G₄₈,K₇₃, E ₁₉₂, and D₂₂₀ which all lie within the catalytic domaincontributed to the naming and uses of hjak2.

The complete nucleic acid sequence encoding hjak2 (SEQ ID NO: 1)disclosed herein, provides the basis for the design of antisensemolecules useful in diminishing or eliminating expression of the genomicnucleotide sequence. For example, hjak2, or its oligonucleotides,fragments, portions, or complement, may be used in diagnostichybridization or amplification assays of biopsied tissue to detectand/or quantify abnormalities in gene expression associated with animmunological disorder. The present invention also relates, in part, tothe inclusion of the nucleic acid sequence in an expression vector whichcan be used to transform host cells or organisms. Such transgenic hostsare useful for production and recovery of the encoded HJAK2.

The invention further comprises using purified HJAK2 polypeptide toproduce antibodies or to identify antagonists or inhibitors which bindHJAK2. Anti-HJAK antibodies may be used in membrane, tissue-based orELISA technologies to detect any disease state or condition related tothe aberrant expression of HJAK2. Antibodies, antagonists or inhibitorscan be used to bind HJAK2 preventing the transfer of high energyphosphate molecules and therefore signal transduction. The inventionalso comprises pharmaceutical compositions containing the peptide,antibodies, antagonists or inhibitors for the diagnosis, prevention ortreatment of conditions associated with altered or uncontrolled hjak2expression. These conditions may include, but are not limited to:arteriosclerosis, asthma, bronchitis, emphysema, inflammatory boweldisease, leukemia, oncogenesis, osteoarthritis, psoriasis, rheumatoidarthritis, septic shock, and systemic lupus erythematosus. Steps fortesting a biological sample with probes, oligomers, fragments orportions of the hjak2 nucleotide sequence or antibodies produced againstthe purified HJAK2 protein are provided.

Antisense molecules, antibodies, antagonists or inhibitors (includingproteins, peptides, oligopeptides or organic molecules capable ofcompromising or modulating HJAK2 expression) may also be used fortherapeutic purposes, for example, in neutralizing the aberrant activityof a HJAK2 associated with, for example, inflammation or oncogenesis.The present invention also provides for pharmaceutical compositions forthe treatment of disease states associated with aberrant expression ofhjak2 comprising the aforementioned antisense molecules, antibodies,antagonists or inhibitors.

DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H display displays an alignmentof the nucleic acid (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequencesof human jak2 kinase. Alignments shown in these and in the followingfigures were produced using the multisequence alignment program ofDNASTAR software (DNASTAR Inc. Madison Wis.).

FIGS. 2A, 2B, 2C, 2D, 2E and 2F show the amino acid sequence similaritybetween HJAK2 (SEQ ID NO:2) and MUSPTK1 (GI 409584; SEQ ID NO:3).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the abbreviation for the novel human Jak2 kinase inlower case (hjak2) refers to a gene, cDNA, RNA, or nucleic acidsequence, while the upper case version (HJAK2) refers to a protein,polypeptide, peptide, oligopeptide, or amino acid sequence.

An “oligonucleotide” or “oligomer”is a stretch of nucleotide residueswhich has a sufficient number of bases to be used in a polymerase chainreaction (PCR). These short sequences are based on (or designed from)genomic or cDNA sequences and are used to amplify, confirm, or revealthe presence of an identical, similar or complementary DNA or RNA in aparticular cell or tissue. Oligonucleotides or oligomers compriseportions of a DNA sequence having at least about 10 nucleotides and asmany as about nucleotides, preferably about 15 to 30 nucleotides. Theyare chemically synthesized and may be used as probes.

“Probes” are nucleic acid sequences of variable length, preferablybetween at least about 10 and as many as about 6,000 nucleotides. Theyare used in the detection of identical, similar, or complementarynucleic acid sequences. Longer length probes are usually obtained from anatural or recombinant source, are highly specific and much slower tohybridize than oligonucleotides. They may be single- or double-strandedand are carefully designed to have specificity in PCR, hybridizationmembrane-based, or ELISA-like technologies.

“Reporter” molecules are chemical moieties used for labelling a nucleicor amino acid sequence. They include, but are not limited to,radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents. Reporter molecules associate with, establish the presence of,and may allow quantification of a particular nucleic or amino acidsequence.

A “portion” or “fragment” of a polynucleotide or nucleic acid comprisesall or any part of the nucleotide sequence having fewer nucleotides thanabout 6 kb, preferably fewer than about 1 kb which can be used as aprobe. Such probes may be labelled with reporter molecules using nicktranslation, Klenow fill-in reaction, PCR or other methods well known inthe art. After pretesting to optimize reaction conditions and toeliminate false positives, nucleic acid probes may be used in Southern,northern or in situ hybridizations to determine whether DNA or RNAencoding the protein is present in a biological sample, cell type,tissue, organ or organism.

“Recombinant nucleotide variants” are polynucleotides which encode aprotein. They may be synthesized by making use of the “redundancy” inthe genetic code. Various codon substitutions, such as the silentchanges which produce specific restriction sites or codon usage-specificmutations, may be introduced to optimize cloning into a plasmid or viralvector or expression in a particular prokaryotic or eukaryotic hostsystem, respectively.

“Linkers” are synthesized palindromic nucleotide sequences which createinternal restriction endonuclease sites for ease of cloning the geneticmaterial of choice into various vectors. “Polylinkers” are engineered toinclude multiple restriction enzyme sites and provide for the use ofboth those enzymes which leave 5′ and 3′ overhangs such as BamHI, EcoRI,PstI, KpnI and Hind III or which provide a blunt end such as EcoRV,SnaBI and StuI.

“Control elements” or “regulatory sequences” are those nontranslatedregions of the gene or DNA such as enhancers, promoters, introns and 3′untranslated regions which interact with cellular proteins to carry outreplication, transcription, and translation. They may occur as boundarysequences or even split the gene. They function at the molecular leveland along with regulatory genes are very important in development,growth, differentiation and aging processes.

“Chimeric” molecules are polynucleotides or polypeptides which arecreated by combining one or more of nucleotide sequences of thisinvention (or their parts) with additional nucleic acid sequence(s).Such combined sequences may be introduced into an appropriate vector andexpressed to give rise to a chimeric polypeptide which may be expectedto be different from the native molecule in one or more of the followingcharacteristics: cellular location, distribution, ligand-bindingaffinities, interchain affinities, degradation/turnover rate,signalling, etc.

“Active” refers to those forms, fragments, or domains of an amino acidsequence which display the biologic and/or immunogenic activitycharacteristic of the naturally occurring peptide.

“Naturally occurring HJAK2” refers to a polypeptide produced by cellswhich have not been genetically engineered or which have beengenetically engineered to produce the same sequence as that naturallyproduced. Specifically contemplated are various polypeptides which arisefrom post-translational modifications. Such modifications of thepolypeptide include but are not limited to acetylation, carboxylation,glycosylation, phosphorylation, lipidation and acylation.

“Derivative”refers to those polypeptides which have been chemicallymodified by such techniques as ubiquitination, labelling (see above),pegylation (derivatization with polyethylene glycol), and chemicalinsertion or substitution of amino acids such as ornithine which do notnormally occur in human proteins.

“Recombinant polypeptide variant” refers to any polypeptide whichdiffers from naturally occurring HJAK2 by amino acid-insertions,deletions and/or substitutions, created using recombinant DNAtechniques. Guidance in determining which amino acid residues may bereplaced, added or deleted without abolishing characteristics ofinterest may be found by comparing the sequence of HJAK2 with that ofrelated polypeptides and minimizing the number of amino acid sequencechanges made in highly conserved regions.

Amino acid “substitutions” are defined as one for one amino acidreplacements. They are conservative in nature when the substituted aminoacid has similar structural and/or chemical properties. Examples ofconservative replacements are substitution of a leucine with anisoleucine or valine, an aspartate with a glutamate, or a threonine witha serine.

Amino acid “insertions” or “deletions” are changes to or within an aminoacid sequence. They typically fall in the range of about 1 to 5 aminoacids. The variation allowed in a particular amino acid sequence may beexperimentally determined by producing the peptide synthetically or bysystematically making insertions, deletions, or substitutions ofnucleotides in the hjak2 sequence using recombinant DNA techniques.

A “signal or leader sequence” is a short amino acid sequence which orcan be used, when desired, to direct the polypeptide through a membraneof a cell. Such a sequence may be naturally present on the polypeptidesof the present invention or provided from heterologous sources byrecombinant DNA techniques.

An “oligopeptide”is a short stretch of amino acid residues and may beexpressed from an oligonucleotide. It may be functionally equivalent toand either the same length as or considerably shorter than a “fragment”,a “portion”, or a “segment” of a polypeptide. Such sequences comprise astretch of amino acid residues of at least about 5 amino acids and oftenabout 17 or more amino acids, typically at least about 9 to 13 aminoacids, and of sufficient length to display biologic and/or immunogenicactivity.

An “inhibitor” is a substance which retards or prevents a chemical orphysiological reaction or response. Common inhibitors include but arenot limited to antisense molecules, antibodies, antagonists and theirderivatives.

A “standard”is a quantitative or qualitative measurement use forcomparison. Preferably, it is based on a statistically appropriatenumber of samples and is created to use as a basis of comparison whenperforming diagnostic assays, running clinical trials, or followingpatient treatment profiles. The samples of a particular standard may benormal or similarly abnormal.

“Animal” as used herein may be defined to include human, domestic (cats,dogs, etc.), agricultural (cows, horses, sheep, goats, chicken, fish,etc.), test species (frogs, mice, rats, rabbits, simians, etc.)

“Conditions” includes cancers, disorders or diseases in which hjak2activity may be implicated. These specifically include, but are notlimited to, anemia, arteriosclerosis, asthma, bronchitis, emphysema,gingivitis, inflammatory bowel disease, insulin-dependent diabetesmellitus leukemia, multiple endocrine neoplasias, osteoarthritis,osteoporosis, pulmonary fibrosis, rheumatoid arthritis, septic shocksyndromes, and systemic lupus erythematosus.

Since the list of technical and scientific terms cannot be allencompassing, any undefined terms shall be construed to have the samemeaning as is commonly understood by one of skill in the art to whichthis invention belongs. Furthermore, the singular forms “a”, “an” and“the”include plural referents unless the context clearly dictatesotherwise. For example, reference to a “restriction enzyme” or a “highfidelity enzyme” may include mixtures of such enzymes and any otherenzymes fitting the stated criteria, or reference to the method includesreference to one or more methods for obtaining cDNA sequences which willbe known to those skilled in the art or will become known to them uponreading this specification.

Before the present sequences, variants, formulations and methods formaking and using the invention are described, it is to be understoodthat the invention is not to be limited only to the particularsequences, variants, formulations or methods described. The sequences,variants, formulations and methodologies may vary, and the terminologyused herein is for the purpose of describing particular embodiments. Theterminology and definitions are not intended to be limiting since thescope of protection will ultimately depend upon the claims.

DESCRIPTION OF THE INVENTION

The present invention provides for a purified polynucleotide whichencodes a novel human Jak2 kinase which is expressed in human cells ortissues. The human Jak2 kinase (hjak2; Incyte Clone 179527) was firstidentified among the cDNAs from a placenta cDNA library. The naming andproscribed uses of the present invention are based in part on theconserved residues found in HJAK2. These particularly include theresidues G₄₈, K₇₃, E₁₉₂, and D₂₂₀, which are all found within thecatalytic domain. Computer search and alignment of the full length aminoacid sequences showed that HJAK2 has 92% similarity to murine Jak2kinase (MUSPTK1; GenBank GI 409584; Wilks A F (1989) Proc Nat Acad Sci86:1603–7), which in turn has 96% sequence similarity with human Jak1kinase.

Purified nucleotide sequences, such as hjak2, have numerous applicationsin techniques known to those skilled in the art of molecular biology.These techniques include their use as PCR or hybridization probes, forchromosome and gene mapping, in the production of sense or antisensenucleic acids, in screening for new therapeutic molecules, etc. Theseexamples are well known and are not intended to be limiting.Furthermore, the nucleotide sequences disclosed herein may be used inmolecular biology techniques that have not yet been developed, providedthe new techniques rely on properties of nucleotide sequences that arecurrently known, including, but not limited to, such properties as thetriplet genetic code and specific base pair interactions.

As a result of the degeneracy of the genetic code, a multitude ofHJAK2-encoding nucleotide sequences may be produced. Some of these willbear only minimal homology to the endogenous sequence of any known andnaturally occurring Jak2 kinase sequence. This invention hasspecifically contemplated each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring HJAK2 and all such variations are to beconsidered as being specifically disclosed.

Although the hjak2 nucleotide sequence and its derivatives or variantsare preferably capable of identifying the nucleotide sequence of thenaturally occurring HJAK2 under optimized conditions, it may beadvantageous to produce HJAK2-encoding nucleotide sequences possessing asubstantially different codon usage. Codons can be selected to increasethe rate at which expression of the peptide occurs in a particularprokaryotic or eukaryotic expression host in accordance with thefrequency with which particular codons are utilized by the host. Otherreasons for substantially altering the nucleotide sequence encoding theHJAK2 without altering the encoded amino acid sequence include theproduction of RNA transcripts having more desirable properties, such asa longer half-life, than transcripts produced from the naturallyoccurring sequence.

Nucleotide sequences encoding HJAK2 may be joined to a variety of 26other nucleotide sequences by means of well established recombinant DNAtechniques (Sambrook J et al (1989) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y., Chapters4, 8, 16, and 17 or Ausubel FM et al (1989) Current Protocols inMolecular Biology, John Wiley & Sons, New York New York N.Y., Chapters9, 13, and 16). Useful sequences for joining to hjak2 include anassortment of cloning vectors such as plasmids, cosmids, lambda phagederivatives, phagemids, and the like. Vectors of interest includevectors for replication, expression, probe generation, sequencing, andthe like. In general, vectors of interest may contain an origin ofreplication functional in at least one organism, convenient restrictionendonuclease sensitive sites, and selectable markers for one or morehost cell systems.

PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188provides additional uses for oligonucleotides based upon the hjak2nucleotide sequence. Such oligomers are generally chemicallysynthesized, but they may be of recombinant origin or a mixture of both.Oligomers generally comprise two nucleotide sequences, one with senseorientation (5′−>3′) and one with antisense orientation (3′ to 5′)employed under optimized conditions for identification of a specificgene or diagnostic use. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for identification and/or quantitation ofclosely related DNA or RNA sequences.

Full length genes may be cloned utilizing partial nucleotide sequenceand various methods known in the art. Sarkar (1993; PCR Methods Applic2:318–22) disclose “restriction-site PCR”as a direct method which usesuniversal primers to retrieve unknown sequence adjacent to a knownlocus. First, genomic DNA is amplified in the presence of primer tolinker and a primer specific to the known region. The amplifiedsequences are subjected to a second round of PCR with the same linkerprimer and another specific primer internal to the first one. Productsof each round of PCR are transcribed with an appropriate RNA polymeraseand sequenced using reverse transcriptase. Sarkar present dataconcerning Factor IX for which they identified a conserved stretch of 20nucleotides in the 3′ noncoding region of the gene.

Inverse PCR is the first method to report successful acquisition ofunknown sequences starting with primers based on a known region (TrigliaT 26 et al (1988) Nucleic Acids Res 16:8186). The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template. Divergent primers are designed from theknown region. The multiple rounds of restriction enzyme digestions andligations that are necessary prior to PCR make the procedure slow andexpensive (Gobinda et al, supra).

Capture PCR (Lagerstrom M et al (1991) PCR Methods Applic 1:111–19) is amethod for PCR amplification of DNA fragments adjacent to a knownsequence in human and YAC DNA. As noted by Sarkar (supra), capture PCRalso requires multiple restriction enzyme digestions and ligations toplace an engineered double-stranded sequence into an unknown portion ofthe DNA molecule before PCR. Although the restriction and ligationreactions are carried out simultaneously, the requirements forextension, immobilization and two rounds of PCR and purification priorto sequencing render the method cumbersome and time consuming.

Parker J D et al (1991; Nucleic Acids Res 19:3055–60), teach walkingPCR, a method for targeted gene walking which permits retrieval ofunknown sequence. In this same vein, PROMOTERFINDER™ a new kit availablefrom Clontech (Palo Alto Calif.) uses PCR and primers derived from p53to walk in genomic DNA. Nested primers and special PromoterFinderlibraries are used to detect upstream sequences such as promoters andregulatory elements. This process avoids the need to screen librariesand is useful in finding intron/exon junctions.

Another new PCR method, “Improved Method for Obtaining Full Length cDNASequences” by Guegler et al, patent application Ser. No. 08/487,112,filed Jun. 7, 1995 and hereby incorporated by reference, employs XL-PCR(Perkin-Elmer, Foster City Calif.) to amplify and extend partialnucleotide sequence into longer pieces of DNA. This method was developedto allow a single researcher to process multiple genes (up to 20 ormore) at one time and to obtain an extended (possibly full-length)sequence within 6–10 days. This new method replaces methods which uselabelled probes to screen plasmid libraries and allow one researcher toprocess only about 3–5 genes in 14–40 days.

In the first step, which can be performed in about two days, any two ofa plurality of primers are designed and synthesized based on a knownpartial sequence. In the second step, which takes about six to eighthours, the sequence is extended by PCR amplification of a selectedlibrary. The third and fourth steps, which take about one day, arepurification of the amplified cDNA and its ligation into an appropriatevector. The fifth step, which takes about one day, involves transformingand growing up host bacteria. The sixth step, which takes approximatelyfive hours, PCR is used to screen bacterial clones for extendedsequence. The final steps, which take about one day, involve thepreparation and sequencing of selected clones.

If the full length cDNA has not been obtained, the entire procedure isrepeated using either the original library or some other preferredlibrary. The preferred library may be one that has been size-selected toinclude only larger cDNAs or may consist of single or combinedcommercially available libraries, eg. lung, liver, heart and brain fromGibco/BRL (Gaithersburg Md.). The cDNA library may have been preparedwith oligo (dT) or random priming. Random primed libraries are preferredin that they will contain more sequences which contain 5′ ends of genes.A randomly primed library may be particularly useful if an oligo (dT)library does not yield a complete gene. It must be noted that the largerand more complex the protein, the less likely it is that the completegene will be found in a single plasmid.

A new method for analyzing either the size or the nucleotide sequence ofPCR products is capillary electrophoresis. Systems for rapid sequencingare available from Perkin Elmer (Foster City Calif.), BeckmanInstruments (Fullerton Calif.), and other companies. Capillarysequencing employs flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/Light intensity is converted to electricalsignal using appropriate software (eg. GENOTYPER™ and SEQUENCENAVIGATORS from Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display is computercontrolled. Capillary electrophoresis provides greater resolution and ismany times faster than standard gel based procedures. It is particularlysuited to the sequencing of small pieces of DNA which might be presentin limited amounts in a particular sample. The reproducible sequencingof up to 350 bp of M13 phage DNA in 30 min has been reported(Ruiz-Martinez MC et al (1993) Anal Chem 65:2851–8).

Another aspect of the subject invention is to provide for hjak2hybridization probes which are capable of hybridizing with naturallyoccurring nucleotide sequences encoding HJAK2. The stringency of thehybridization conditions will determine whether the probe identifiesonly the native nucleotide sequence of hjak2 or sequences of otherclosely related Jak2 kinase molecules. If degenerate hjak2 nucleotidesequences of the subject invention are used for the detection of relatedkinase encoding sequences, they should preferably contain at least 50%of the nucleotides of the sequences presented herein. Hybridizationprobes of the subject invention may be derived from the nucleotidesequence presented in SEQ ID NO: 1 or from surrounding genomic sequencescomprising untranslated regions such as promoters, enhancers andintrons. Such hybridization probes may be labelled with appropriatereporter molecules.

Means for producing specific hybridization probes for this Jak2 kinaseinclude oligolabelling, nick translation, end-labelling or PCRamplification using a labelled nucleotide. Alternatively, the cDNAsequence may be cloned into a vector for the production of an mRNAprobe.

Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by addition of an appropriateRNA polymerase such as T7, T3 or SP6 and labeled nucleotides. A numberof companies (such as Pharmacia Biotech, Piscataway N.J.; Promega,Madison Wis.; US Biochemical Corp, Cleveland, Ohio; etc.) supplycommercial kits and protocols for these procedures.

It is also possible to produce a DNA sequence, or portions thereof,entirely by synthetic chemistry. Sometimes the source of information forproducing this sequence comes from the known homologous sequence fromclosely related organisms. After synthesis, the nucleic acid sequencecan be used alone or joined with a pre-existing sequence and insertedinto one of the many available DNA vectors and their respective hostcells using techniques well known in the art. Moreover, syntheticchemistry may be used to introduce specific mutations into thenucleotide sequence. Alternatively, a portion of sequence in which amutation is desired can be synthesized and recombined with a portion ofan existing genomic or recombinant sequence.

Hjak2 nucleotide sequence can be used in a diagnostic test or assay todetect disorder or disease processes associated with abnormal expressionof hjak2. The nucleotide sequence is added to a sample (fluid, cell ortissue) from a patient under hybridizing conditions. After an incubationperiod, the sample is washed with a compatible fluid which optionallycontains a reporter molecule which will bind the specific nucleotide.After the compatible fluid is rinsed off, the reporter molecule isquantitated and compared with a standard for that fluid, cell or tissue.If hjak2 expression is significantly different from the standard, theassay indicates the presence of disorder or disease. The form of suchqualitative or quantitative methods may include northern analysis, dotblot or other membrane-based technologies, dip stick, pin or chiptechnologies, PCR, ELISAs or other multiple sample format technologies.

This same assay, combining a sample with the nucleotide sequence, isapplicable in evaluating the efficacy of a particular therapeutictreatment regime. It may be used in animal studies, in clinical trials,or in monitoring the treatment of an individual patient. First, standardexpression must be established for use as a basis of comparison. Second,samples from the animals or patients affected by a disorder or diseaseare combined with the nucleotide sequence to evaluate the deviation fromthe standard or normal profile. Third, an entirely new or pre-existingtherapeutic agent is administered, and a treatment profile is generated.This post-treatment assay is evaluated to determine whether the patientprofile progresses toward or returns to the standard pattern. Successivetreatment profiles may be used to show the efficacy of treatment over aperiod of several days or several months.

The nucleotide sequence for hjak2 can also be used to generate probesfor mapping native genomic sequence. The sequence may be mapped to aparticular chromosome or to a specific region of the chromosome usingwell known techniques. These include in situ hybridization tochromosomal spreads (Verma et al (1988) Human Chromosomes: A Manual ofBasic Techniques, Pergamon Press, New York City), flow-sortedchromosomal preparations, or artificial chromosome constructions such asyeast artificial chromosomes (YACs), bacterial artificial chromosomes(BACs), bacterial P1 constructions or single chromosome cDNA libraries.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers are invaluable in extending genetic maps. Examples of suchgenetic maps can regularly be found in the journal Science (eg, 1994;265:1981f). Often the placement of a gene on the chromosome of anothermammalian species may reveal associated markers even if the number orarm of a particular human chromosome is not known. New sequences can beassigned to chromosomal arms, or parts thereof, by physical mapping.This provides valuable information to investigators searching fordisease genes using positional cloning or other gene discoverytechniques. Once a disease or syndrome, such as ataxia telangiectasia(AT), has been crudely localized by genetic linkage to a particulargenomic region, for example, AT to 11q22–23 (Gatti et al (1988) Nature336:577–580), any sequences mapping to that area may representassociated or regulatory genes for further investigation. The nucleotidesequence of the subject invention may also be used to detect differencesin the chromosomal location due to translocation, inversion, etc.between normal and carrier or affected individuals.

The nucleotide sequence encoding HJAK2 may be used to produce an aminoacid sequence using well known methods of recombinant DNA technology.Goeddel (1990, Gene Expression Technology. Methods and Enzymology, Vol185, Academic Press, San Diego Calif.) is one among many publicationswhich teach expression of an isolated, purified nucleotide sequence. Theamino acid or peptide may be expressed in a variety of host cells,either prokaryotic or eukaryotic. Host cells may be from the samespecies from which the nucleotide sequence was derived or from adifferent species. Advantages of producing an amino acid sequence orpeptide by recombinant DNA technology include obtaining adequate amountsfor purification and the availability of simplified purificationprocedures.

Cells transformed with hjak2 nucleotide sequence may be cultured underconditions suitable for the expression and recovery of peptide from cellculture. The peptide produced by a recombinant cell may be secreted ormay be contained intracellularly depending on the sequence and/or thevector used. In general, it is more convenient to prepare recombinantproteins in secreted form, and this is accomplished by ligating hjak2 toa recombinant nucleotide sequence which directs its movement through aparticular prokaryotic or eukaryotic cell membrane. Other recombinantconstructions may join hjak2 to nucleotide sequence encoding apolypeptide domain which will facilitate protein purification (Kroll D Jet al (1993) DNA Cell Biol 12:441–53).

Direct peptide synthesis using solid-phase techniques (Creighton (1983)Proteins Structures And Molecular Principles, W H Freeman and Co, NewYork N.Y. pp. 50–60) is an alternative to recombinant or chimericpeptide production. Automated synthesis may be achieved, for example, anApplied Biosystems 431A peptide synthesizer in accordance with theinstructions provided by the manufacturer. Additionally HJAK2 or anypart thereof may be mutated during direct synthesis and combined usingchemical methods with other kinase sequences, or parts thereof.

Although an amino acid sequence or oligopeptide used for antibodyinduction does not require biological activity, it must be immunogenic.HJAK2 used to induce specific antibodies may have an amino acid sequenceconsisting of at least five amino acids and preferably at least 10 aminoacids. Short stretches of amino acid sequence may be fused with those ofanother protein such as keyhole limpet hemocyanin, and the chimericpeptide used for antibody production. Alternatively, the peptide may beof sufficient length to contain an entire domain.

Antibodies specific for HJAK2 may be produced by inoculation of anappropriate animal with an antigenic fragment of the peptide. Anantibody is specific for HJAK2 if it is produced against an epitope ofthe polypeptide and binds to at least part of the natural or recombinantprotein. Antibody production includes not only the stimulation of animmune response by injection into animals, but also analogous processessuch as the production of synthetic antibodies, the screening ofrecombinant immunoglobulin libraries for specific-binding molecules(Orlandi R et al (1989) PNAS 86:3833–3837, or Huse WD et al (1989)Science 256:1275–1281), or the in vitro stimulation of lymphocytepopulations. Current technology (Winter G and Milstein C (1991) Nature349:293–299) provides for a number of highly specific binding reagentsbased on the principles of antibody formation. These techniques may beadapted to produce molecules which specifically bind HJAK2. Antibodiesor other appropriate molecules generated against a specific immunogenicpeptide fragment or oligopeptide can be used in Western analysis,enzyme-linked immunosorbent assays (ELISA) or similar tests to establishthe presence of or to quantitate amounts of HJAK2 active in normal,diseased, or therapeutically treated cells or tissues.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention.

EXAMPLES

I Placenta cDNA Library Construction

The cDNA library was constructed from normal placenta. The tissue waslysed in a buffer containing guanidinium isothiocyanate. The lysate wasextracted with phenol chloroform and precipitated with ethanol. Poly A+RNA was isolated using biotinylated oligo d(T) primer and streptavidincoupled to a paramagnetic particle (Promega Corp. Madison Wis.) and sentto Stratagene (La Jolla Calif.) for cDNA library preparation. The cDNAsynthesis was primed using both oligo d(T) and random hexamers, and thetwo cDNA libraries were treated separately. Synthetic adapteroligonucleotides were ligated onto the ends of the cDNAs which weredigested with XhoI and inserted into the UNIZAP vector system(Stratagene).

The PBLUESCRIPT phagemid (Stratagene) was excised from each library, andphagemids from the two cDNA libraries were combined into a singlelibrary by mixing equal numbers of bacteriophage. The phagemids weretransformed into E. coli host strain XLI-BLUE (Stratagene). Enzymes fromboth PBLUESCRIPT and a cotransformed f1 helper phage nicked the DNA,initiated new DNA synthesis, and created the smaller, single-strandedcircular plasmid DNA molecules which contained the cDNA insert. Theplasmid DNA was released, purified, and used to reinfect fresh hostcells (SOLR, Stratagene). Presence of the β-lactamase gene on theplasmid allowed transformed bacteria to grow on medium containingampicillin.

II Isolation of cDNA Clones

Plasmid DNAs containing the cDNA insert were purified using theQIAWELL-8 plasmid purification system from QIAGEN Inc (ChatsworthCalif.) according to standard protocol. The DNA was eluted and preparedfor DNA sequencing and other analytical manipulations.

The cDNA inserts from random isolates of the placenta library werepartially sequenced. The cDNAs were sequenced by the method of Sanger Fand AR Coulson (1975; J Mol Biol 94:441f), using a CATALYST 800 or aMACROLAB 2200 (Hamilton, Reno Nev.) in combination with four Peltierthermal cyclers (PTC200 from MJ Research, Watertown Mass.) and AppliedBiosystems 377 or 373 DNA sequencing systems (Perkin Elmer), and readingframe was determined.

III Sequencing of cDNA Clones

The cDNA inserts from random isolates of the placenta library weresequenced in part. Methods for DNA sequencing are well known in the artand employ such enzymes as the Klenow fragment of DNA polymerase I,SEQUENASE® (US Biochemical Corp., or Taq polymerase. Methods to extendthe DNA from an oligonucleotide primer annealed to the DNA template ofinterest have been developed for both single- and double-strandedtemplates. Chain termination reaction products were separated usingelectrophoresis and detected via their incorporated, labelledprecursors. Recent improvements in mechanized reaction preparation,sequencing and analysis have permitted expansion in the number ofsequences that can be determined per day. Preferably, the process isautomated with machines such as the MICROLAB 2200 (Hamilton, Reno Nev.),Peltier thermal cycler (PTC200; MJ Research, Watertown Mass.) and theApplied Biosystems Catalyst 800 and 377 and 373 DNA sequencers.

The quality of any particular cDNA library may be determined byperforming a pilot scale analysis of the cDNAs and checking forpercentages of clones containing vector, lambda or E. coli DNA,mitochondrial or repetitive DNA, and clones with exact or homologousmatches to public databases. The number of unique sequences, thosehaving no known match in any available database, are then recorded.

IV Homology Searching of cDNA Clones and Their Deduced Proteins

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems and incorporated intothe INHERIT 670 sequence analysis system. In this algorithm, PatternSpecification Language (TRW Inc, Los Angeles Calif.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches.

Alternatively, BLAST, which stands for Basic Local Alignment SearchTool, is used to search for local sequence alignments (Altschul SF(1993) J Mol Evol 36:290–300; Altschul, SF et al (1990) J Mol Biol215:403–10). BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. While it is useful for matches whichdo not contain gaps, it is inappropriate for performing motif-stylesearching. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

The partial hjak2 molecule presented and claimed in this application wasidentified using the criteria above. The full length nucleic and aminoacid sequences for this novel human Jak2 kinase are shown in FIGS. 1A,1B, 1C, 1D, 1E, 1F, 1G, and 1H.

FIGS. 2A, 2B, 2C, 2D, and 2E shows the alignment between the translatedamino acid sequence for hjak2 and the closest related molecule, murineJak2 kinase (MUSPTK1; GenBank GI 409584; Wilks AF (1989) Proc Nat AcadSci 86:1603–7).

V Extension of cDNAs to Full Length

The partial sequence originally identified in Incyte Clone 179527 wasused to design oligonucleotide primers for extension of the cDNAs tofull length. Primers are designed based on known sequence; one primer issynthesized to initiate extension in the antisense direction (XLF) andthe other to extend sequence in the sense direction (XLF). The primersallow the sequence to be extended “outward” generating ampliconscontaining new, unknown nucleotide sequence for the gene of interest.The primers may be designed using OLIGO 4.0 (National Biosciences Inc,Plymouth Minn.), or another appropriate program, to be 22–30 nucleotidesin length, to have a GC content of 50% or more, and to anneal to thetarget sequence at temperatures about 68°–72° C. Any stretch ofnucleotides which would result in hairpin structures and primer—primerdimerizations was avoided.

The placenta cDNA library was used with “XLR=GGGCGGAAGTGCTCTCGGCGGAAG”with --XLR (SEQ ID NO:4) and “XLF=AGTGTGCTACAGTGCTGGTCGTCG” with --XLF(SEQ ID NO:5) primers to extend and amplify Incyte Clone 179527 toobtain the full length Jak2 kinase sequence.

By following the instructions for the XL-PCR kit and thoroughly mixingthe enzyme and reaction mix, high fidelity amplification is obtained.Beginning with 40 pmol of each primer and the recommended concentrationsof all other components of the kit, PCR is performed using the Peltierthermal cycler (PTC200; MJ Research, Watertown Mass.) and the followingparameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat step 4–6 for 15 additional cyclesStep 8 94° C. for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15min Step 11 Repeat step 8–10 for 12 cycles Step 12 72° C. for 8 min Step13 4° C. (and holding)

A 5–10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6–0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Although allextensions potentially contain a full length gene, some of the largestproducts or bands are selected and cut out of the gel. Furtherpurification involves using a commercial gel extraction method such asQIAQUICK (QIAGEN Inc). After recovery of the DNA, Klenow enzyme is usedto trim single-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer. Then, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2–3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook J et al, supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook J et al, supra)containing 2×Carb. The following day, 12 colonies are randomly pickedfrom each plate and cultured in 150 μl of liquid LB/2×Carb medium placedin an individual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture is transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample is transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2–4 for an additional29 cycles Step 6 72° C. for 180 sec Step 7 40° C. (and holding)

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid and sequenced.

VI Diagnostic Assay Using Hjak2 Specific Oligomers

In those cases where a specific condition (see definitions, supra) issuspected to involve expression of altered quantities of hjak2,oligomers may be designed to establish the presence and/or quantity ofmRNA expressed in a biological sample. There are several methodscurrently being used to quantitate the expression of a particularmolecule. Most of these methods use radiolabelled (Melby P C et al 1993J Immunol Methods 159:235–44) or biotinylated (Duplaa C et al 1993 AnalBiochem 212:229–236) nucleotides, coamplification of a control nucleicacid, and standard curves onto which the experimental results areinterpolated. Quantitation may be speeded up by running the assay in anELISA format where the oligomer-of-interest is presented in variousdilutions and a colorimetric response gives rapid quantitation. Forexample, a complete HJAK2 deficiency may result in the inability toundergo cell division or to react to an infectious organism. In likemanner, overexpression may cause major inflammation, swelling and majortissue damage. In either case, a quick diagnosis may allow healthprofessionals to treat the condition and prevent worsening of thecondition. This same assay can be used to monitor progress of thepatient as his/her physiological situation moves toward the normal rangeduring therapy.

VII Sense or Antisense Molecules

Knowledge of the correct cDNA sequence of this Jak2 kinase or itsregulatory elements enable its use as a tool in sense (Youssoufian H andH F Lodish 1993) Mol Cell Biol 13:98–104) or antisense (Eguchi et al(1991) Annu Rev Biochem 60:631–652) technologies for the investigationor alteration of gene expression. To inhibit in vivo or in vitro hjak2expression, an oligonucleotide based on the coding sequence of an hjak2designed with OLIGO 4.0 software (National Biosciences Inc) is used.Alternatively, a fragment of an hjak2 is produced by digesting hjak2coding sequence with restriction enzymes. These enzymes and specificrestrictions sites may be selected using INHERIT analysis software(Applied Biosystems), and the strands separated by heating the fragmentsand selecting for the antisense strand. Either the oligonucleotide orthe fragment may be used to inhibit hjak2 expression. Furthermore,antisense molecules can be designed to inhibit promoter binding in theupstream nontranslated leader or at various sites along the hjak2 codingregion. Alternatively, antisense molecules may be designed to inhibittranslation of an mRNA into polypeptide by preparing an oligomer orfragment which will bind in the region spanning approximately −10 to +10nucleotides at the 5′ end of the coding sequence. These technologies arenow well known to those of in the art.

In addition to using antisense molecules constructed to interrupttranscription of the open reading frame, modifications of geneexpression can be obtained by designing antisense sequences toenhancers, introns, or even to trans-acting regulatory genes. Similarly,inhibition can be achieved using Hogeboom base-pairing methodology, alsoknown as “triple helix” base pairing. Triple helix pairing compromisesthe ability of the double helix to open sufficiently for the binding ofpolymerases, transcription factors, or regulatory molecules.

Any of these types of antisense molecules may be placed in expressionvectors and used to transform preferred cells or tissues. This mayinclude introduction of the expression vector into a organ, tumor,synovial cavity or the vascular system for transient or short termtherapy or introduction via gene therapy technologies for long termtreatment. Transient expression may last for a month or more with anon-replicating vector and three months or more if appropriatereplication elements are used in the transformation or expressionsystem.

Stable transformation of appropriate dividing cells with a vectorcontaining the antisense molecule can produce a transgenic cell line,tissue or organism (see, for example, Trends in Biotechnol 11:155–215(1993) and U.S. Pat. No. 4,736,866, 12 Apr. 1988). Those cells whichassimilate or replicate enough copies of the vector to allow stableintegration will also produce enough antisense molecules to compromiseor entirely eliminate normal activity of the hjak2. Frequently, thefunction of an hjak2 can be ascertained by observing behaviors such aslethality, loss of a physiological pathway, changes in morphology, etc.at the cellular, tissue or organismal level.

VIII Expression of HJAK2

Expression of the HJAK2 may be accomplished by subcloning the cDNA intoappropriate vectors and transfecting the vectors into host cells. Inthis case, the cloning vector previously used for the generation of thetissue library also provides for direct expression of the hjak2 sequencein E. coli. Upstream of the cloning site, this vector contains apromoter for β-galactosidase, followed by sequence containing theamino-terminal Met and the subsequent 7 residues of β-galactosidase.Immediately following these eight residues is a bacteriophage promoteruseful for transcription and a linker containing a number of uniquerestriction sites.

Induction of an isolated, transfected bacterial strain with IPTG usingstandard methods will produce a fusion protein corresponding to thefirst seven residues of β-galactosidase, about 5 to 15 residues whichcorrespond to linker, and the peptide encoded within the hjak2 cDNA.Since cDNA clone inserts are generated by an essentially random process,there is one chance in three that the included cDNA will lie in thecorrect frame for proper translation. If the cDNA is not in the properreading frame, it can be obtained by deletion or insertion of theappropriate number of bases by well known methods including in vitromutagenesis, digestion with exonuclease III or mung bean nuclease, oroligonucleotide linker inclusion.

The cDNA can be shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonucleotide linkerscontaining cloning sites as well as a stretch of DNA sufficient tohybridize to the end of the target cDNA (25 bases) can be synthesizedchemically by standard methods. These primers can then used to amplifythe desired gene fragments by PCR. The resulting fragments can bedigested with appropriate restriction enzymes under standard conditionsand isolated by gel electrophoresis. Alternatively, similar genefragments can be produced by digestion of the cDNA with appropriaterestriction enzymes and filling in the missing gene sequence withchemically synthesized oligonucleotides. Partial nucleotide sequencefrom more than one kinase homolog can be ligated together and clonedinto appropriate vectors to optimize expression.

Suitable expression hosts for such chimeric molecules include but arenot limited to mammalian cells such as Chinese Hamster Ovary (CHO) andhuman 293 cells, insect cells such as Sf9 cells, yeast cells such asSaccharomyces cerevisiae, and bacteria such as E. coli. For each ofthese cell systems, a useful expression vector may also include anorigin of replication to allow propagation in bacteria and a selectablemarker such as the β-lactamase antibiotic resistance gene to allowselection in bacteria. In addition, the vectors may include a secondselectable marker such as the neomycin phosphotransferase gene to allowselection in transfected eukaryotic host cells. Vectors for use ineukaryotic expression hosts may require RNA processing elements such as3′ polyadenylation sequences if such are not part of the cDNA ofinterest.

If native promoters are not part of the cDNA, other host specificpromoters may be specifically combined with the coding region of hjak2.They include MMTV, SV40, and metallothionein promoters for CHO cells;trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor,alcohol oxidase and PGH promoters for yeast. In addition, transcriptionenhancers, such as the rous sarcoma virus (RSV) enhancer, may be used inmammalian host cells. Once homogeneous cultures of recombinant cells areobtained through standard culture methods, large quantities ofrecombinantly produced peptide can be recovered from the conditionedmedium and analyzed using methods known in the art.

Ix Isolation of Recombinant HJAK2

HJAK2 may be expressed as a recombinant protein with one or moreadditional polypeptide domains added to facilitate protein purification.

Such purification facilitating domains include, but are not limited to,metal chelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, SeattleWash.). The inclusion of a cleavable linker sequence such as Factor XAor enterokinase (Invitrogen) between the purification domain and thehjak2 sequence may be useful to facilitate expression of HJAK2.

X Testing HJAK2 Activity

The sequence for HJAK2 in this application present many differentdomains (and subdomains as detailed in the background of the invention)which may be utilized: 1) individually for the production of antibodies,2) in functional groups (eg. to span a membrane), and 3) asinterchangeable, usable parts of a chimeric kinase. For example, aknown, full length kinase such as the hjak2 kinase of this applicationmay be used to swap related portions of the nucleic acid sequence,analogous to domains or subdomains of MAP kinase polypeptides. Thechimeric nucleotides, so produced, may be introduced into prokaryotichost cells (as reviewed in Strosberg A D and Marullo S (1992) TrendsPharma Sci 13:95–98) or eukaryotic host cells. These host cells are thenemployed in procedures to determine what molecules activate the kinaseor what molecules are activated by a kinase. Such activating oractivated molecules may be of extracellular, intracellular, biologic orchemical origin.

An example of a test system, in this case for hjak2 kinase, can be basedon the interaction of protein tyrosine kinases with chemokine receptors(Taniguchi T (1995) Science 268:251–255). These receptors are capable ofactivating a variety of nonreceptor protein tyrosine kinases whenstimulated by an extracellular chemokine. C—X—C chemokines such asplatelet factor 4, interleukin-8, connective tissue activating proteinIII, neutrophil activating peptide 2, are soluble activators ofneutrophils.

A standard measure of neutrophil activation involves measuring themobilization of Ca⁺⁺ as part of the signal transduction pathway. Theexperiment involves several steps. First, blood cells obtained fromvenipuncture are fractionated by centrifugation on density gradients.Enriched populations of neutrophils are further fractionated on columnsby negative selection using antibodies specific for other blood cellstypes. Next, neutrophils are transformed with an expression vectorcontaining the kinase nucleic acid sequence of interest and preloadedfluorescent probe whose emission characteristics have been altered byCa⁺⁺ binding. Or in the alternative, the neutrophil is preloaded withthe purified kinase of interest and fluorescent probe. Then, when thecells are exposed to an appropriate chemokine, the chemokine receptoractivates the kinase which, in turn, initiates Ca⁺⁺ flux. Ca⁺⁺mobilization is observed and measured using fluorometry as has beendescribed in Grynkievicz G et al (1985) J Biol Chem 260:3440, and McCollS et al (1993) J Immunol 150:4550–4555, incorporated herein byreference.

XI Identification of or Production of HJAK2 Specific Antibodies

Purified HJAK2 is used to screen a pre-existing antibody library or toraise antibodies using either polyclonal or monoclonal methodology. In apolyclonal approach, denatured protein from the reverse phase HPLCseparation is obtained in quantities up to 75 mg. This denatured proteincan be used to immunize mice or rabbits using standard protocols; about100 micrograms are adequate for immunization of a mouse, while up to 1mg might be used to immunize a rabbit. For identifying mouse hybridomas,the denatured protein can be radioiodinated and used to screen potentialmurine B-cell hybridomas for those which produce antibody. Thisprocedure requires only small quantities of protein, such that 20 mgwould be sufficient for labeling and screening of several thousandclones.

In a monoclonal approach, the amino acid sequence of HJAK2, as deducedfrom translation of the cDNA, is analyzed to determine regions of highimmunogenicity. Oligopeptides comprising appropriate hydrophilicregions, as shown in FIG. 3, are synthesized and used in suitableimmunization protocols to raise antibodies. Analysis to selectappropriate epitopes is described by Ausubel FM et al (supra). Theoptimal amino acid sequences for immunization are usually at theC-terminus, the N-terminus and those intervening, hydrophilic regions ofthe polypeptide which are likely to be exposed to the externalenvironment when the protein is in its natural conformation.

Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH,Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS; Ausubel FM et al, supra). If necessary, a cysteine may beintroduced at the N-terminus of the peptide to permit coupling to KLH.Rabbits are immunized with the peptide-KLH complex in complete Freund'sadjuvant. The resulting antisera are tested for antipeptide activity bybinding the peptide to plastic, blocking with 1% BSA, reacting withantisera, washing and reacting with labeled (radioactive orfluorescent), affinity purified, specific goat anti-rabbit IgG.

Hybridomas may also be prepared and screened using standard techniques.Hybridomas of interest are detected by screening with labeled HJAK2 toidentify those fusions producing the monoclonal antibody with thedesired specificity. In a typical protocol, wells of plates (FAST;Becton-Dickinson, Palo Alto, Calif.) are coated with affinity purified,specific rabbit-anti-mouse antibodies (or suitable anti-species Ig) at10 mg/ml. The coated wells are blocked with 1% BSA, washed and exposedto supernatants from hybridomas. After incubation the wells are exposedto labeled HJAK2, 1 mg/ml. Clones producing antibodies will bind aquantity of labeled HJAK2 which is detectable above background. Suchclones are expanded and subjected to 2 cycles of cloning at limitingdilution (1 cell/3 wells). Cloned hybridomas are injected into pristinemice to produce ascites, and monoclonal antibody is purified from mouseascitic fluid by affinity chromatography on Protein A. Monoclonalantibodies with affinities of at least 10⁸/M, preferably 10⁹ to 10¹⁰ orstronger, will typically be made by standard procedures as described inHarlow and Lane (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor N.Y.; and in Goding (1986)Monoclonal Antibodies: Principles and Practice, Academic Press, New YorkCity, both incorporated herein by reference.

XII Diagnostic Test Using HJAK2 Specific Antibodies

Particular HJAK2 antibodies are useful for the diagnosis ofprepathologic conditions, and chronic or acute diseases which arecharacterized by differences in the amount or distribution of HJAK2. Todate, HJAK2 has been found only in the placenta library; however, itsactivity there is most probably associated with organ function,inflammation or defense.

Diagnostic tests for HJAK2 include methods utilizing the antibody and alabel to detect HJAK2 in human body fluids, tissues or extracts of suchtissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a reporter molecule. A wide variety of labels andconjugation techniques are known and have been reported extensively inboth the scientific and patent literature. Suitable reporter moleculesor labels include those radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents previously mentioned as well assubstrates, cofactors, inhibitors, magnetic particles and the like.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced as shown inU.S. Pat. No. 4,816,567, incorporated herein by reference.

A variety of protocols for measuring soluble or membrane-bound HJAK2,using either polyclonal or monoclonal antibodies specific for therespective protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HJAK2 is preferred, but a competitive binding assay may be employed.These assays are described, among other places, in Maddox, D E et al(1983, J Exp Med 158:1211).

XIII Purification of Native HJAK2 Using Specific Antibodies

Native or recombinant HJAK2 can be purified by immunoaffinitychromatography using antibodies specific for that particular HJAK2. Ingeneral, an immunoaffinity column is constructed by covalently couplingthe anti-HJAK2 antibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia Biotech). Likewise, monoclonal antibodies areprepared from mouse ascites fluid by ammonium sulfate precipitation orchromatography on immobilized Protein A. Partially purifiedimmunoglobulin is covalently attached to a chromatographic resin such asCNBr-activated SEPHAROSE (Pharmacia Biotech). The antibody is coupled tothe resin, the resin is blocked, and the derivative resin is washedaccording to the manufacturer's instructions.

Such immunoaffinity columns may be utilized in the purification of HJAK2by preparing a fraction from cells containing HJAK2 in a soluble-form.This preparation may be derived by solubilization of whole cells or of asubcellular fraction obtained via differential centrifugation (with orwithout addition of detergent) or by other methods well known in theart. Alternatively, soluble HJAK2 containing a signal sequence may besecreted in useful quantity into the medium in which the cells aregrown.

A soluble HJAK2-containing preparation is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of HJAK2 (eg, high ionic strength buffers in thepresence of detergent). Then, the column is eluted under conditions thatdisrupt antibody/HJAK2 binding (eg, a buffer of pH 2–3 or a highconcentration of a chaotrope such as urea or thiocyanate ion), and HJAK2is collected.

XIV Drug Screening

This invention is particularly useful for screening therapeuticcompounds by using binding fragments of HJAK2 in any of a variety ofdrug screening techniques. The peptide fragment employed in such a testmay either be free in solution, affixed to a solid support, borne on acell surface or located intracellularly. One may measure, for example,the formation of complexes between HJAK2 and the agent being tested.

Alternatively, one can examine the diminution in complex formationbetween HJAK2 and a receptor caused by the agent being tested.

Methods of screening for drugs or any other agents which can affectmacrophage activation comprise contacting such an agent with HJAK2fragment and assaying for the presence of a complex between the agentand the HJAK2 fragment. In such assays, the HJAK2 fragment is typicallylabelled. After suitable incubation, free HJAK2 fragment is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular agent to bind toHJAK2.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the HJAK2 polypeptidesand is described in detail in European Patent Application 84/03564,published on Sep. 13, 1984, incorporated herein by reference. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with HJAK2 fragment andwashed. Bound HJAK2 fragment is then detected by methods well known inthe art. Purified HJAK2 can also be coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding HJAK2specifically compete with a test compound for binding to HJAK2fragments. In this manner, the antibodies can be used to detect thepresence of any peptide which shares one or more antigenic determinantswith HJAK2.

XV Identification of Molecules Which Interact with HJAK2

The inventive purified HJAK2 is a research tool for identification,characterization and purification of interacting molecules. Appropriatelabels are incorporated into HJAK2 by various methods known in the artand HJAK2 is used to capture soluble or interact with membrane-boundmolecules.

A preferred method involves labeling the primary amino groups in HJAK2with ¹²⁵I Bolton-Hunter reagent (Bolton, AE and Hunter, W M (1973)Biochem J 133: 529). This reagent has been used to label variousmolecules without concomitant loss of biological activity (Hebert Calif.et al (1991) J Biol Chem 266: 18989–94; McColl S et al (1993) J Immunol150:4550–4555). Membrane-bound molecules are incubated with the labelledHJAK2 molecules, washed to removed unbound molecules, and the HJAK2complex is quantified. Data obtained using different concentrations ofHJAK2 are used to calculate values for the number, affinity, andassociation of HJAK2.

Labelled HJAK2 fragments are also useful as a reagent for thepurification of molecules with which HJAK2 interacts, specificallyincluding inhibitors. In one embodiment of affinity purification, HJAK2is covalently coupled to a chromatography column. Cells and theirmembranes are extracted, HJAK2 is removed and various HJAK2-freesubcomponents are passed over the column. Molecules bind to the columnby virtue of their HJAK2 affinity. The HJAK2-complex is recovered fromthe column, dissociated and the recovered molecule is subjected toN-terminal protein sequencing or other identification procedure. If thecaptured molecule has an amino acid sequence, it can be used to designdegenerate oligomers for use in cloning the gene from an appropriatecDNA library.

In an alternate method, monoclonal antibodies raised against HJAK2fragments are screened to identify those which inhibit the binding oflabelled HJAK2. These monoclonal antibodies are then used in affinitypurification or expression cloning of associated molecules. Othersoluble binding molecules are identified in a similar manner. LabelledHJAK2 is incubated with extracts or other appropriate materials derivedfrom lung, kidney or other tissues with activated monocytes ormacrophages. After incubation, HJAK2 complexes (which are larger thanthe lone HJAK2 fragment) are identified by a sizing technique such assize exclusion chromatography or density gradient centrifugation and arepurified by methods well known in the art. The soluble bindingprotein(s) are subjected to N-terminal sequencing to obtain informationsufficient for database identification, if the soluble protein is known,or for cloning, if the soluble protein is unknown.

XVI Use and Administration of Antibodies or Inhibitors to HJAK2

The antibodies and inhibitors can provide different effects whenadministered therapeutically. The antibodies and inhibitors are used tolessen or eliminate undue damage caused by disorders or diseasesassociated with upregulated HJAK2 expression. Each of these molecules ortreatments (TSTs) will be formulated in a nontoxic, inert,pharmaceutically acceptable aqueous carrier medium preferably at a pH ofabout 5 to 8, more preferably 6 to 8, although the pH may vary accordingto the different characteristics of the peptide, antibody or inhibitorbeing formulated and the condition to be treated. Characteristics ofTSTs include solubility of the molecule, half-life,antigenicity/immunogenicity and the ability of the inhibitor to reachits target(s). These and other characteristics may aid in defining aneffective carrier. Native human proteins are preferred as TSTs, butrecombinant peptides as well as organic or synthetic molecules resultingfrom drug screens may be equally effective in particular situations.

TSTs may be delivered by known routes of administration including butnot limited to topical creams and gels; transmucosal spray and aerosol;transdermal patch and bandage; injectable, intravenous and lavageformulations; and orally administered liquids and pills particularlyformulated to resist stomach acid and enzymes. The particularformulation, exact dosage, and route of administration will bedetermined by the attending physician and will vary according to eachspecific situation. Such determinations are made by considering multiplevariables such as the condition to be treated, the TST to beadministered, and the pharmacokinetic profile of the particular TST.Additional factors which may be taken into account include disease state(eg. severity) of the patient, age, weight, gender, diet, time andfrequency of administration, drug combination, reaction sensitivities,and tolerance/response to therapy. Long acting TST formulations might beadministered every 3 to 4 days, every week; or once every two weeksdepending on half-life and clearance rate of the particular TST.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature. See U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Those skilled in the art will employ different formulations fordifferent TSTs. Administration to lung cells may necessitate delivery ina manner different from that to kidney or other cells.

It is contemplated that conditions associated with altered HJAK2expression are treatable with TSTs. These conditions, which specificallyinclude, but are not limited to, anemia, arteriosclerosis, asthma,bronchitis, emphysema, gingivitis, inflammatory bowel disease,insulin-dependent diabetes mellitus, leukemia, multiple endocrineneoplasias, osteoarthritis, osteoporosis, pulmonary fibrosis, rheumatoidarthritis, septic shock syndromes, and systemic lupus erythematosus maybe specifically diagnosed by the tests discussed above. In addition,such tests may be used to monitor treatment.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

1. An isolated polynucleotide encoding a polypeptide selected from thegroup consisting of: a) the amino acid sequence of SEQ ID NO:2, and b) afragment of the amino acid sequence of SEQ ID NO:2, wherein saidfragment has kinase activity.
 2. An isolated polynucleotide of claim 1which encodes a polypeptide comprising the amino acid sequence of SEQ IDNO:2.
 3. An isolated polynucleotide of claim 1 which encodes apolypeptide comprising a fragment of an amino acid sequence of SEQ IDNO:2, wherein said fragment has kinase activity.
 4. A recombinantpolynucleotide comprising a promoter sequence operably linked to apolynucleotide of claim
 1. 5. An isolated transformed with a recombinantpolynucleotide of claim
 4. 6. A method for producing a polypeptideencoded by the polynucleotide of claim 1, the method comprising: a)culturing a cell under conditions suitable for expression of thepolypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide of claim 1, and b)recovering the polypeptide so expressed.